Pain is often complicated by clinical comorbidities such as anxiety and depression. Most preclinical models, however, do not examine the impact of such comorbidities on pain. For example, while epidemiological studies have consistently shown a comorbid relationship between pain and anxiety, the impact of anxiety on the transition from acute to chronic pain remains unknown largely due to the lack of preclinical models. In an effort to establish a preclinical model of combined pain and anxiety, we recently demonstrated in a set of preliminary experiments that anxiety-like behavior (elevated plus maze, dark-light box test) was induced in rats with persistent, but not transient, nociception from peripheral nerve injury. The presence of anxiety-like behavior prolonged and exacerbated nociceptive behavior. Furthermore, the expression of neuropeptide S (NPS, a novel neuropeptide with an endogenous anxiolytic property) was downregulated, associated with increased basal and stimulation-evoked neural activities (fMRI), in the amygdala of those rats exhibiting nociceptive and anxiety-like behavior. Intracerebroventricular administration of exogenous NPS concurrently improved nociceptive and anxiety-like behavior in the same rats. These preliminary results demonstrate a significant impact of anxiety-like behavior on the transition to chronic pain and suggest that the central NPS system may play a critical role in the comorbid interaction between pain and anxiety. In this grant application, we propose to establish a rat model of combined nociceptive and anxiety-like behavior and use this model to a) evaluate contributions of anxiety-like behavior during the transition from acute to chronic pain and b) determine the role of the central NPS system in the comorbid interaction between pain and anxiety. We will achieve this goal by using behavioral and pharmacological tools, immunohistochemistry, Western blot, real-time PCR, in situ hybridization, cell culture, ELISA, patch-clamp recording, and rodent functional MRI (fMRI). In Specific Aim 1, we will evaluate the impact of nociception-induced vs. genetically pre-disposed anxiety on nociceptive behavior and brain neural activities (rodent fMRI) following transient (subcutaneous carrageenan injection) or persistent (mental nerve injury) nociception in rats. In Specific Aim 2, we will examine a functional role for the central NPS system in nociceptive and anxiety-like behavior using NPS and NPSR (NPS receptor) knockout mice. In Specific Aim 3, we will investigate the neural and cellular mechanism underlying the interaction between pain and anxiety by determining the effect of NPS on a) neural activities in the brain regions implicated in nociceptive processing and emotional/affective response (rodent fMRI) and b) an intra-amygdaloid NPS-GABA link contributory to the regulation of anxiety disorder (patch-clamp recording and cell culture). We anticipate that this project will a) establish a preclinical model useful to examine the impact of anxiety on the transition from acute to chronic pain and b) suggest a strategy for the concurrent treatment of pain and anxiety by regulating the central NPS system.